1. Field of the Invention
This invention relates to the field of nucleic acid transfection, and more particularly to methods for preparing precipitated complexes of calcium phosphate and nucleic acid, called transfectacons herein, and methods for nucleic acid transfection of eukaryotic cells by calcium phosphate co-precipitation.
2. Related Disclosures
The ability to introduce foreign DNA into eukaryotic host cells is one of the principal tools of recombinant DNA technology. Methods for transfecting eukaryotic host cells with foreign DNA can be broadly grouped into four categories: (1) direct introduction of cloned DNA by microinjection or microparticle bombardment; (2) use of viral vectors; (3) encapsulation within a carrier system; and (4) use of transfecting reagents such as calcium phosphate and diethylaminoethyl (DEAE)-dextran.
Several attempts have been made to improve the transient transfection of mammalian cells using a variety of transfecting reagents that include cationic lipids, DEAE-dextran (or its related analogs), and calcium phosphate (Itani et al., Gene, 56: 267-276 (1987); Hofland et al., Proc. Natl. Acad. Sci. (USA), 93: 7305-7309 (1996); Smyth-Templeton et al., Nature Biotechnology, 15: 647-652 (1997); McCutchman and Pagano, J. Natl. Cancer Inst., 41: 351 (1968); Parker and Stark, J. Virol., 31: 360 (1979); Graham et al., Nature (Lond.), 251: 687-691 (1974); Bachetti and Graham, Proc. Natl. Acad. Sci. (USA), 74(4): 1590-1594 (1977); Wigler et al., Cell, 14: 725-731 (1978)). Of the reagents used as facilitators of DNA transfection, calcium phosphate remains the most widely used because of its simplicity and general effectiveness for a wide variety of cell types. Hence, significant attention has been given to improvement of transient transfections using calcium-phosphate/DNA (CaPi/DNA) particles as the transfecting reagent. Hereinafter, the complex formed between a transfecting reagent such as CaPi and the plasmid or nucleic acid that is being introduced into the host cell of choice is referred to as a transfectacon.
The process of introducing nucleic acid sequences into mammalian cells via CaPi transfectacons was first described by Graham and van der Eb, Virology, 52: 456-467 (1973). This method, which was modified by Wigler et al., Cell, 14: 725-731 (1978) and by Chen and Okayama, Mol. Cell. Biol., 7: 2745-2752 (1987), is based on the formation of small insoluble CaPi transfectacons that attach to the cell surface and are subsequently transported into the cytoplasm via endocytosis (Loyter et al., Proc. Natl. Acad. Sci. (USA), 79: 422-426 (1982); Loyter et al., Exp. Cell Res., 139: 223-234 (1982)). Once internalized, these nucleic acids are transported to the nucleus by means of an endosomal-lysosomal vesicular transport system (Orrantia et al., Somat. Cell Mol. Gen., 16: 305-310 (1990); Orrantia et al., Exper. Cell Res., 190: 170-174 (1990); Coonrod et al., Gene Therapy, 4: 1313-1321 (1997)).
The most widely used protocol for CaPi/DNA transient transfection of mammalian cells (Sambrook et al., Molecular Cloning: A Laboratory Manual 2nd Ed. (Cold Spring Harbor Laboratory Press: Cold Spring Harbor, N.Y., 1989)) involves the formation of CaPi transfectacons at pH 7.05 and standard concentrations of CaCl.sub.2, Na.sub.2 HPO.sub.4, and DNA, which are incubated for 20-30 minutes at room temperature. Although this protocol is easily implemented in most laboratories for the generation of both stable and transient transfections, there are several problems that are experienced when using this method (Sambrook et al., supra): 1) this protocol is highly variable from transfection to transfection; 2) scaling this process is very difficult due to the lack of specifications for the variables in the reactions; and 3) the titers achieved are low compared to other transfection systems.
In an attempt to improve CaPi transfections of mammalian cells, particular attention has been given to various aspects of the transfectacon formation both as a separate step prior to its addition to cells and as a simultaneous step wherein the transfectacons are formed in the presence of adherent cells or suspension-adapted cells (Song and Lahiri, Nucleic Acids Res., 23(17): 3609-3611 (1995); Jordan et al., Nucleic Acids Res., 24 (4): 596-601 (1996); Jordan et al., Cytotechnology, 26: 39-47 (1998)). The formation of transfection-competent CaPi transfectacons has been shown to be sensitive to changes in pH of less than 0.1 pH units (Chen and Okayama, supra; O'Mahoney and Adams, DNA Cell Biol., 13: 1227-1232 (1994); Jordan et al., Nucleic Acids Res., supra). The basis of this sensitivity is not completely understood, but a recent report suggests that the pH of the precipitation reaction affects the flocculation coefficient and the zeta potential for the CaPi transfectacons (Yang and Yang, Drug Delivery, 3: 173-179 (1996); Yang and Yang, Drug Delivery, 3: 181-186 (1996)), under the standard concentrations for the reactants used in these studies.
In other investigations designed to examine the significance of the concentrations of the reactants in the co-precipitation reactions, improvements to the existing protocol were achieved (Chen and Okayama, supra; Song and Lahiri, supra; Jordan et al., Nucleic Acids Res., supra; Wilson et al., Anal. Biol. 226: 212-220 (1995)). In these experiments, one variable was changed between each experiment while the remaining variables were held constant, which makes assessment of the interactions amongst variables very difficult.
Many investigations into the length of time required for improving co-precipitation of CaPi transfectacons have been conducted. These studies show that at standard or near-standard concentrations of calcium, phosphate, and DNA, shorter precipitation times have resulted in higher transfection efficiencies and/or expression titers relative to transfections that followed the standard protocol (O'Mahoney and Adams, supra; Jordan et al., Nucleic Acids Res., supra; Coonrod et al., supra). It has been proposed that these shorter precipitation times yield CaPi transfectacons that are more easily taken up by cells, presumably due to smaller particle size of the precipitate. See also U.S. Pat. Nos. 5,633,156; 5,593,875; 5,686,263; and 5,484,720, which describe the incubation of particles so that they grow to an average length of up to about 300 nm. To date, accurate measurement of these particles and correlation between particle size and transfection efficiency or protein expression have not been reported (Parasrampuria, BioPharm., 3:38-45 (1998)).
Additionally, throughout the literature, standard or near standard co-precipitation conditions are not very robust in that they yield highly variable titers between transfections that are performed on different days (Sambrook et al., supra).
Since the original and modified protocols yield relatively low transfection efficiencies and expression in experiments geared towards transient or stable transfections, there is still a need for an improved and robust method of calcium phosphate transfection and improved titers for recombinant proteins. In addition, there is a need for methods of calcium phosphate transfection in suspension culture, particularly in the area of large-scale suspension culture, which is currently lacking.